Molecular Mechanisms Of Aging Effects Of C.elegans And Health Risks Induced By Tris(1,3-dichloro-2-propyl) Phosphate

Tris(1,3-dichloro-2-propyl)phosphate(TDCPP)is a halogen organophosphorus compound As an additive flame retardant,it is used in many consumer products and is one of the important substitutes for polybrominated diphenyl ethers(PBDEs).At present,TDCPP and its main diester metabolite bis(1,3-dichloro-2-propyl)phosphate(BDCPP)have been widely detected in a variety of environmental media and organisms,even in human breast milk,plasma,placenta,semen and urine samples,posing a serious threat to the ecological environment and human health.Several studies have confirmed that exposure to TDCPP pollution may cause severe degenerative diseases such as nerve damage,growth retardation and reproductive ability decline.Thus it is probably to induce aging effect and reduce the lifespan of organisms.However,the aging effect and molecular mechanisms induced by TDCPP exposure is still limited,and it is unable to explain the reason of degenerative diseases caused by TDCPP in organisms completely.Therefore,it is needed to further study the aging effect and mechanisms in organisms following TDCPP exposure,to reveal ecological toxicology of TDCPP and provide a scientific basis for the damage of human health mechanism.Thus,this study selected Caenorhabditis elegans(C.elegans)as a model for aging research,extracted the scientific problems on TDCPP inducing aging effect and molecular mechanism,researched toxicological effect on C.elegans and the aging mechanism caused by TDCPP exposure systemly.This study analysed physiological and biochemical characteristics changes,genes and proteins expression differences by adopting the method of multidisciplinary cross analysis;identified the target genes,proteins,biomarkers and pivotal signaling pathway significantly related to TDCPP-accelerated aging process by using transcriptomics,bioinformatics,transgenic and mutation strains comprehensively;combined with in vitro human cell experiments,homology modeling and molecular docking analysis,explored the mechanisms of TDCPP on crucial homologous proteins and signaling pathways between different species including human,zebrafish and nematode to reveal the potential health risks of chlorinated organophosphate esters(ClOPEs)TDCPP exposure.The main conclusions of the present study were as following:(1)TDCPP exposure damaged locomotion behaviors and reduce lifespan to accelerate the aging process of C.elegans.Through multiple scenarios(acute and subacute)exposure to TDCPP at different concentrations(control,0.1,1,100 and 1000 μg/L)to evaluation the physiological and biochemical toxicity of C.elegans,this study found at same concentration,subacute exposure(L1-72 h)is more sensitive than acute exposure.Exposure to TDCPP at environmental concentrations reduced the body length,brood size,locomotion behaviors and lifespan of C.elegans,as well as increase the intestinal permeability,oxidative stress,and the level of apoptosis and lipofuscin.Among them,after subacute exposure,the 10%effect concentration(EC10)of head thrash and body bend frequency which positively correlated with lifespan were only 11.5 and 39.4 μg/L,respectively,which were in the range of environmental concentration;In addition,subacute exposure to TDCPP at 1000 μg/L reduce the average lifespan of nematodes by 17%,increased the lipofuscin level by 32%significantly,and increased the oxidative stress level by 34%significantly,indicating TDCPP reduce the lifespan of nematodes in a dose-dependent manner and cause aging-related degenerative diseases.(2)TDCPP accelerated aging process of C.elegans by oxidative free radical damage mechanism.TDCPP exposure induces the nematodes to produce excessive reactive oxide species(ROS),and further reacts with lipids to amplify the oxidative damage,leading to increased lipofuscin levels and crucial biomarkers of aging,4-hydroxynon-2-enal(4-HNE)and other key products of lipid peroxidation(LPO)accumulation in the nematodes,which ultimately induces degenerative diseases and reduce lifespan of C.elegans.In addition,this study through mRNA transcriptome sequencing technology(mRNA-seq)and bioinformatics to evaluate overall mRNA expression characteristics and enriched signaling pathways,it was found that glutathione metabolism and glutathione S-transferase gene(gst-5,gst-6,gst-9,gst-10,gst-19,gst-24,gst-26,gst-29,gst-33,gst-38)play an important role in the antioxidant process of C.elegans,further indicating that lipids peroxidation accumulation and oxidative free radical damage mechanisms play a key role in accelerating the aging process of nematodes.(3)TDCPP accelerated aging process and decrease longevity of C.elegans by triggering unconventional Insulin/Insulin-like Growth factor-1(Insulin/IGF-1)signaling pathway.This pathway is not routinely by disrupting the insulin like growth factor-1 receptor DAF-2/IGF1R as previously reported,but by directly inhibiting the downstream tumor-suppressor factor DAF-18/PTEN.This inhibition reduces the second messenger PI(3,4,5)P3 dephosphorylation,activates downstream serine/threonine protein kinase Akt/PKB,and further induces DAF-16/FoxO phosphorylation,inhibits the expression of DAF-16/FoxO and promotes the sequestration of DAF-16/FoxO in the cytoplasm,ultimately reducing the locomotion behaviors and lifespan of nematodes.(4)TDCPP exposure induced specific microRNA(miRNA)to inhibit DAF-16/FoxO to reduce the lifespan of nematodes.Overall evaluation of miRNA expression characteristics and enriched signaling pathway by Small RNA sequencing(Small RNA-seq),it is found that TDCPP exposure could also trigger the unconventional Insulin/IGF-1 pathway,which was highly consistent with the results of mRNA research.Therefore,this study established a small interaction network diagram of miRNA-mRNA that TDCPP reducing lifespan.In addition,miRNA mutant strain studies have shown that miR-48 and miR-84 in let-7 family inhibit DAF-16/FoxO transcription and play a key role in locomotion behavior and lifespan related to aging.Meanwhile,the results of modeling and molecular docking showed the binding mode and affinity between TDCPP and miR-48/84 from the perspective of three-dimensional structure,proving that TDCPP could inhibit the function of DAF-16/FoxO in the unconventional Insulin/IGF-1 pathway by disturbing the expression of miR-48 and miR-84 in C.elegans and ultimately affect the lifespan.(5)The unconventional Insulin/IGF-1 pathway activated by TDCPP exposure has a high degree of homology between species.The unconventional Insulin/IGF-1 signaling pathway has been verified in L02 cells,and crucial homologous genes and proteins expressions in this pathway were consistent with the results in nematodes,confirming the signaling pathways is highly homologous in human cells.In addition,the 3D structure of IGF1R and PTEN proteins in human,zebrafish and nematode was constructed using homology modeling technology,and the molecular docking of TDCPP with multi-species IGF1R or PTEN proteins was studied.The results of specific docking sites analysis proved the key role of tumor suppressor PTEN in the process of aging of C.elegans induced by TDCPP exposure.In summary,TDCPP at environmental concentration could accelerate the aging process and reduce the lifespan of C.elegans.The aging effects mainly through the oxidative free radical damage mechanism,unconventional Insulin/IGF-1 pathway,specific miRNAs(miR-48/84)inhibit DAF-16/FoxO mechanism.Among them,the unconventional Insulin/IGF-1 pathway mechanism has a high degree of homology between different species.This study is helpful to understand the toxic effect and mechanism of ClOPEs at a deeper level and provide a scientific basis for the establishment of ecological toxicology and health risk warning as well as prevention and control measures of environmental ClOPEs pollution.